Biological Sciences and Pharmaceutical Research Vol.1 (2), pp. 022-029, October 2013 Available online at http://www.journalissues.org/IBSPR/ © 2013 Journal Issues ISSN 2350-1588

Original Research Paper

Synthesis and antimicrobial evaluation of some chalcones

Accepted 21 October, 2013

Haruna Baba*1, Odigwe Azubike2 and Cyril O.

Usifoh2

1Department of Pharmaceutical and Medicinal Chemistry, Faculty

of Pharmacy, Niger Delta University, Bayelsa State, Nigeria.

2Department of Pharmaceutical Chemistry, Faculty of Pharmacy,

University of Benin, Benin City, Edo State, Nigeria.

*Corresponding Author.

E-mail : babharun@yahoo.co.uk Tel.: +2347034402435

Chalcones are ubiquitous in nature and they are precursors to the flavonoids family. Natural occurring chalcones as well as synthetic chalcone analogues have demonstrated many pharmaceutical effects, including anti-inflammatory, anti-oxidant, anti-parasite, and anti-tumor activities . The synthesis of some substituted chalcones was carried out in alcoholic potassium hydroxide at room temperature by Claisen-Schmidt condensation reaction. The compounds were obtained in good yield and were characterized using the combination of IR and NMR spectroscopy. The antibacterial activity of the synthesized compounds was carried out and they were found to be inactive against the tested strains of micro-organisms. Contrary to the popular belief that most chalcones do exhibit antimicrobial activity, these substituted chalcones lack such activity. Key words: Chalcones, antibacterial activity, micro-organism

INTRODUCTION There has been an increased interest for the development of new and effective antimicrobial drugs as a result of emerging infectious disease and microbial resistance to the available drugs. Chalcones (1,3-diphenyl-propene-1-one) pertaining to the flavonoid family (Yau-Hung et al.,2013) both natural as well as synthetic products have been revised for their wide biological activities such as antibacterial (Alcaraz et al., 2000), anti-tumor (Echeverria et al., 2009, Fang et al.,2013), anti-inflammatory (Beom-Tae et al.,2008),antioxidant (Babasaheb et al.,2010). Various substituted chalcones have been synthesized from substituted benzaldehyde and acetophenone derivatives (Jing Zhang et al.,2013) and some have been found to have inherent pharmacological activity or could be used as starting materials in the synthesis of various other compounds such as some sulphonilamides , anthocyanins e.g. flavanones, flavones isoflavones etc. As part of our continued search for effective antimicrobial agents, three substituted chalcones; 1-(3,4,5-trimethoxyphenyl)-3-(3’,4’,5’-trimethoxyphenyl)-2-propen-1-one, 1-(3, 4, 5-trimethoxyphenyl)-3-(2, 4, 6-trimethoxyphenyl)-2-propen-1-one and 1-(3, 4, 5 -trimethoxyphenyl)-3-(2’, 4’-dimethoxyphenyl)-2-propen-1-one were synthesized and investigated for possible antibacterial activity.

MATERIALS AND METHODS Chemistry Melting points were taken in an open capillary tube using Galenkap equipment and are uncorrected. IR spectrum was recorded on a Buck Scientific IR M500 instrument (Buck Scientific Inc, Norwalk, Connecticut, USA). NMR spectrum was recorded on a Varian Gemini 200 (250 MHz) (Varian Inc, Palo Alto, California, USA). Chemical shifts are reported as δ in ppm relative to tetramethylsilane (TMS). All the analytical grade reagents and solvents used were obtained from commercial sources and used without further purification. A number of chalcone derivatives were prepared by a Claisen-Schmidt condensation (Funiss et al., 2004) of the appropriate aldehyde and acetophenone derivatives (Figure 1). General Procedure for the synthesis of chalcones: a solution of substituted acetophenone (6mM) and aromatic aldehyde (6mM) in methanol (15mL) was cooled to 5-10°C in an ice bath. The cooled solution was treated with adding a small portion of pulverized potassium hydroxide (10mM). The reaction mixture was magnetically stirred for 60 minutes and then left overnight, monitored by thin layer chromatography using developing solvent n-

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C H 3 R

O +

H

O R '

R

O

R ' 1 . K O H / M e O H , r t

2 . H C l 1 0 %

Figure 1 . C l a i s e n - S c h m i d t c o n d e n s a t i o n . ( 1 ) K O H ( 1 . 5 e q . ) s u b s t t u t e d a c e t o p h e n o n e ( 1 e q . ) . s u b s t i t u t e d b e n z a l d e h y d e ( 1 e q . ) ; M e O H , r o o m t e m p e r a t u r e ; ( 2 ) 1 0 % H C l s o l u t i o n

R : 3 , 4 , 5 , T r i m e t h o x y a c e t o p h e n o n e ; R ' : O C H 3 a t 3 ' , 4 ' , 5 ' ( c o m p d 1 ) , 2 ' , 4 ' , 6 ' ( c o m p d 2 ) , 2 ' , 4 ' ( c o m p d 3 )

Figure 1.Claisen-Schmidt condensation(1)KOH(1.5eq.)substituted acetophenone (1eq.). Substituted benzaldehyde(1eq.);MeOH, room temperature ;(2)10%HCL solution

hexane – acetone (5:1). The resulting dark solution was diluted with ice water and carefully acidified using dilute hydrochloric acid. The chalcone which separated as a yellow solid was collected by filtration after washing with water and further purified by crystallization from methanol. Antimicrobial activity Four clinical bacterial isolates (Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli) were obtained from the microbial bank of Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Benin, Benn city, Nigeria; for the antimicrobial screening of the synthesized compounds. The bacterial isolates were standardized using colony suspension method and matching the strain’s suspension with 0.5 McFarland standard to give a resultant concentration of 1.5 x 108 cfu/ml. The antibiotic susceptibility testing was determined using the modified Kirby-Bauer diffusion technique by swabbing the Mueller–Hinton agar (MHA) (Oxoids U.K) plates with the resultant saline suspension of each strain and four wells were made in the agar with the aid of cork borer (the diameter of the borer is 6mm). The wells were sealed at the bottom with molten sterilized agar; 0.1ml, 0.05ml, 0.025ml, 0.0125ml solutions of the synthesized compounds representing 1000µg/ml, 500µg/ml, 250µg/ml and 125µg/ml respectively were aseptically dispensed into the labeled wells while antibiotic disc (ciprofloxacin 5µg) used as control was placed on the agar aseptically. The plates were then incubated at 37°C for 24 hours. The zone diameters of inhibition produced by each concentration of the compounds and that of the antibiotic discs were measured and recorded (CLSI, 2008). RESULTS Chemical data: Compound 1 1-(3, 4, 5-trimethoxyphenyl)-3-(3’, 4’, 5’-trimethoxyphenyl) -2-propen-1-one

Yield: 0.9671 g (50.02%), Melting point: 129-131°C, IR(KBr): 3010,2840,1652,1577,1500,1458 cm-1 1H NMR(CDCl3): δ(ppm); 7.7 (d, J = 15.52 Hz, 1H), 7.3 (d, J = 15.71 Hz, 1H), 7.2 (s, 2H), 6.8 (s, 2H), 3.91(s, 6H), 3.9 (s, 3H), 3.89 (s, 6H), 3.87 (s, 3H). 13C NMR (CDCl3): δ(ppm); 189.3, 153.5,153.2,144.8,142.7,140.7, 133.6, 130.4, 121.3,106.4,106.0, 60.9, 56.5, 56.3, 56.2. MS: m/z (%); 389[M++1](23), 388[M+](100),387(12), 374(14), 373(65),358(11), 357(48), 345(12),328(5), 313(6), 296(7). The IR, 1H NMR and 13C NMR spectra of the compound are shown in Figure 2, 3, 4 respectively.

Compound 2

1-(3, 4, 5-trimethoxyphenyl)-3-(2’,4’, 6’-trimethoxyphenyl)-2-propen-1-one

Yield: 0.9053 g (46.84%), Melting point: 125-128°C, IR(KBr): 3020, 2840, 1643, 1560,1500,1457cm-1.

1H NMR (CDCl3): δ (ppm); 8.2 (d, J = 15.99 Hz, 1H), 7.8 (d, J = 15.82 Hz, 1H), 7.2 (s, 2H), 6.1 (s, 2H), 3.9 (s, 6H), 3.88 (s, 3H), 3.84 (s, 6H), 3.81 (s, 3H). 13C NMR (CDCl3): δ (ppm); 190.9, 163.1, 161.6, 152.9, 141.8, 135.9, 134.6, 121.9, 106.5, 106.1, 90.6, 60.8, 56.2, 55.7, 55.3. MS: m/z (%); 389[M++1](1), 388[M+](4), 358(22), 357(100), 341(3), 327(5), 296(12), 268(1), 267(1). The IR, 1H NMR and 13C NMR spectra are shown in Figure 5,6, 7 respectively.

Compound 3

1-(3, 4, 5 -trimethoxyphenyl)-3-(2’, 4’-dimethoxyphenyl)-2-propen-1-one.

Yield: 1.3450g (75.12%), melting point: 139-142°C, IR (KBr): 3095, 2840,1646, 1570, 1499, 1458cm-1. 1H NMR (CDCl3): δ(ppm); 8.0 (d, J = 15.61 Hz, 1H), 7.6 (d, J = 8.76 Hz, 1H), 7.4 (d, J = 15.80 Hz, 1H), 6.5 (d, J = 8.76 Hz, 1H), 6.4 (s, 1H), 3.9 (s, 9H), 3.8 (s, 3H). 13C NMR (CDCl3): δ (ppm); 189.8, 163.0, 160.3, 153.0, 140.2, 134.1, 130.6, 120.2, 117.1, 106.1, 105.5, 98.4, 60.8, 56.2, 55.5, 55.4. MS: m/z (%); 359[M++1] (2), 358[M+](9), 357(1), 343(6), 328(21), 327(100),311(2), 297(4),266(9). The IR, 1H NMR and 13C spectra are shown in Figure 8,9,10 respectively.

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Figure 2. IR spectrum of compound I

Figure 3. 1H NMR of compound I

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Figure 4. 13C NMR of compound I

Figure 5. IR Spectrum of compound II

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Figure 6. 1H NMR of compound II

Figure 7. 13C NMR of compound II

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Figure 8. IR spectrum of compound III

Figure 9. 1H NMR of compound III

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Figure 10. 13C NMR of compound III

Table 1. Result of the antibacterial screening

Compound/Concent-ration (mcg/ml) Staphylococcus Aureus Bacillus subtilis Escherichia coli Pseudomonas aeruginosa Compound 1 1000 NI NI NI NI 500 NI NI NI NI 250 NI NI NI NI 125 NI NI NI NI Compound 2 1000 NI NI NI NI 500 NI NI NI NI 250 NI NI NI NI 125 NI NI NI NI Compound 3 1000 NI NI NI NI 500 NI NI NI NI 250 NI NI NI NI 125 NI NI NI NI ciprofloxacin 5µg 20 20 24 16

NI: no inhibition

DISCUSSION Three substituted chalcones; 1-(3,4,5-trimethoxyphenyl)-3-(3’,4’,5’-trimethoxyphenyl)-2-propen-1-one, 1-(3, 4, 5-trimethoxyphenyl)-3-(2, 4, 6-trimethoxyphenyl)-2-propen-1-one and 1-(3, 4, 5 -trimethoxyphenyl)-3-(2’, 4’-dimethoxyphenyl)-2-propen-1-one, were synthesized by Claisen-Schmidt condensation and characterized using the combination of IR, 1H NMR, 13C NMR spectroscopy, and MS. The compounds were obtained in an appreciable yield (46 –

75%) and purity. The diagnostic two protons of the ketoethylenic group of the chalcones appeared at about 7.7 and 7.3 ppm in all the NMR spectra of the compounds. The aromatic protons of the two phenyl groups were seen between 6.8 to 7.2 ppm relative to tetramethylsilane (TMS). The in-vitro antibacterial screening of the synthesized compounds was carried out by agar well diffusion method and it was found that the compounds were inactive against the tested strains of microorganism (Table 1). The reason for this inactivity cannot be immediately proffered because

Issues Biol. Sci. Pharm. Res. 029 it is a common knowledge that chalcones do exhibit reasonable anti-microbial activity. It has been reported that chalcones with methoxy substitution on the B ring do not have antimicrobial activity (Thanh-Dao et al., 2012). This corroborate the findings in this study. Conclusion Three new substituted chalcone derivatives were synthesized. The structures of the synthesized compounds were established by IR, 1H NMR, 13C NMR and MS data. All the synthesized compounds were screened for their in-vitro antibacterial activity against gram-positive and gram-negative bacteria as compared with the standard. In summary, preliminary results indicate that none of the newly synthesized title compounds exhibited promising antibacterial activities. REFERENCES Alcaraz LE, Blanco SE, Puig ON, Tomás F, Ferretti FH.

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Baba H, Azubike O, Usifoh CO (2013).Synthesis and antimicrobial evaluation of some chalcones. Issues Biol. Sci. Pharm. Res. 1(2):022-029.